Use of chondroitin sulphate E (CS-E) for the treatment of diseases or conditions related to collagen fibril formation

ABSTRACT

The present invention comprises the use of chondroitin sulphate (CS-E) or an active fragment thereof for the treatment of diseases or conditions related to collagen fibril formation. Said compounds can be administrated either by oral, topical, injectable or by any other suitable route.

TECHNICAL BACKGROUND

Many proteins of the extracellular matrix (ECM) are modifiedpost-translationally by addition of oligosaccharide chains and are thusknown as glycoproteins. The oligosaccharides are linked eitherO-glycosidically to serine or threonine residues, or N-glycosidically toan asparagine residue. Proteoglycans are glycoproteins that aresubstituted with a particular class of carbohydrate polymers, known asthe glycosaminoglycans (GAGs). Proteoglycans are found in the ECM, atthe cell surface and intracellularly in storage granules. In the ECMthey contribute to the structure and organisation, and at the cellsurface often function as receptors and/or co-receptors. Allglycosaminoglycans (with the exception of hyaluronan) are synthesised ona core protein acceptor, and they are thus an integral component ofproteoglycans (Wight et al., 1981; Heinegård and Paulsson, 1984,review).

Glycosaminoglycans (GAGs) are named to indicate that one of themonosaccharides in the repeating sequence of disaccharides is an aminosugar. The other monosaccharide is an uronic acid (glucuronic acid oriduroic acid), with the exception of keratan sulphate where it is agalactose. While other oligosaccharide substituents may be branched, GAGchains are linear (again, with the exception of keratan sulphate).Proteoglycans may be substituted with one (e.g. decorin) and up to someone hundred (e.g. aggrecan) GAG chains.

There are 4 types of glycosaminoglycans: hyaluronic acid, chondroitinsulphate/dennatan sulphate, heparan sulphate/heparin and keratansulphate. The disaccharides in all glycosaminoglycan chains excepthyaluronan are sulphated, increasing their negative charge and leadingto an extended conformation of the chain. The molecule will occupy largesolvent domains, observed as a high viscosity of a solution. Thisproperty is essential in cartilage and is the basis on which thetissue's resistance lies.

The repeating disaccharide sequence in CS is glucuronicacid-N-acetyl-galactosamine (GlcA-GalNAc), see FIG. 1. Chondroitinsulphate is found in several forms, named chondroitin-4 sulphate, -6sulphate and -D and -E respectively. These forms differ in thesulphation of saccharides. CS-E is a highly sulphated species, which isattached to perlecan in the I and V domains.

FIG. 1. Basic structure of Chondroitin sulfate. Repeating dimeric ullitsof GlcA β1-3 GalNAc. All hydroxy positions may be sulfated or/andepimerised.

The various positions open for sulfatation are numbered.

Chondroitin sulphate/dermatan sulphate is found in all extracellularmatrices. Cartilage and invertebral disc are the tissues richest inchondroitin sulphate (Wight et al., 1981, review). Chondroitin sulphateis synthesised by specific enzymes located in the Golgi. The polymersare assembled onto a linker tri-saccharide. The hydroxyl group of serineresidues followed by a glycine in the protein is substituted with axylose and two successive galactose residues. Thereafter alternatingmonosaccharides of glucuronic acid and N-acetylgalactosamine are addedsuccessively to form the chain. Some glucuronate residues are convertedto iduronate by an epimerase and sulfation is the last event just priorto secretion (Wight et al., 1981, review). In cartilage aggrecan, amember of the family hyalectins, is a chondroitin sulphate proteoglycanand is substituted with some one hundred CS chain and some thirtykeratan sulphate chains. Aggrecan molecules are clustered along HAstrands bound via their N-terminal globular domain. A protein known aslink protein contacts both the HA-binding G1 domain of the aggrecanmolecule and HA, and stabilises the complex. In this manner hundreds ofaggrecan molecules are joined at one end to the HA. Thus, in cartilagematrix chondroitin sulphate is by far the most abundant GAG.

Perlecan was first identified as a large heparan sulphate proteoglycanisolated from the Engelbrecht-Holm-Swarm (EHS) murine basement membranetumour. In basement membranes, it has been shown to bind severaldifferent classes of molecules. In each instance the core protein, theheparan sulphate (HS) side chains or both in concert, are involved inmediating the interaction. The proteoglycan binds to extracellularmatrix components integral to basement membrane such as collagen IV,nidogen, laminin, and fibronectin (Timpl, R. and Brown, J. C. (1996)Bioassays 18, 123-132). Perlecan has also been shown to bindextracellular matrix components outside the basement membrane, e.g.PRELP and collagen type I (Bengtsson, E., Mörgelin, M., Sasaki, T.,Timpl, R., Heinegård, D., and Aspberg, A. (2002) J. Biol. Chem).Perlecan supports cell-attachment both by binding and clusteringintegrins (Brown, J. C., Sasaki, T., Gohring, W., Yamada, Y., and Timpl,R. (1997) Eur. J Biochem. 250, 39-46). Binding to growth factors hasbeen shown for both the HS side-chains (FGF-2 (Aviezer, D., Hecht, D.,Safran, M., Eisinger, M., David, G., and Yayon, A. (1994) Cell 79,1005-1013)) and the core protein (progranulin, (Gonzalez, E. M.,Mongiat, M., Slater, S. J., Baffa, R., and Jozzo, R. V. (2003) J BiolChem)). Based on its interactions, perlecan is assumed to have a role inbasement membrane integrity.

Perlecan was originally thought to be substituted with HS exclusively,but later studies revealed that it is also present in a variantpartially substituted with chondroitin sulphate (CS) (Couchman, J. R.,Kapoor, R., Sthanam, M., and Wu, R. R. (1996) J Biol Chem 271,9595-9602). Both the HS- and the HS/CS-substituted variants of perlecanhave been found in tissues other than basement membrane, for examplecartilage.

The generation of perlecan null mice revealed two particularlyintriguing findings (Arikawa-Hirasawa, E., Watanabe, H., Takani, H.,Hassell, J. R., and Yamada, Y. (1999) Nat Genet. 23, 354-358; Costell,M., Gustafsson, E., Aszódi, A., Morgelin, M., Bloch, W., Hunziker, E.,Addicks, K., Timpl, R., and Fässler, R. (1999) J Cell Biol 147,1109-1122). First, though mice lacking perlecan did develop gravedisorders caused by compromised basement membrane strength or integrity(e.g. rupture of pericardial sac), the initial assembly of basementmembranes seemed to be without complication. The second striking findingwas the severe skeletal defects exhibited, apparently caused by the lackof perlecan in cartilage.

Following the publication of these results at least two human hereditarydiseases with skeletal deficiencies have been ascribed to an underlyingscarcity or complete lack of perlecan, underscoring the relevance ofthis finding in the mouse model (Nicole, S., Davoine, C. S., Topaloglu,H., Cattolico, L., Barral, D., Beighton, P., Hamida, C. B., Hammouda,H., Cruaud, C., White, P. S., Samson, D., Urtizberea, J. A.,Lehmann-Horn, F., Weissenbach, J., Hentati, F., and Fontaine, B. (2000)Nat Genet. 26, 480-483; Arikawa-Hirasawa, E., Wilcox, W. R., Le, A. H.,Silverman, N., Govindraj, P., Hassell, J. R., and Yamada, Y. (2001) NatGenet. 27, 431-434).

In skeletal development, the deposition of a cartilaginous templateprecedes the formation of bones. The integrity of this template is aprerequisite for proper assembly of the skeleton. Perlecan-null mousecartilage shows fewer and less organised collagen type II fibrils, anddecreased levels of aggrecan, indicating a failure to organise theextracellular matrix (Costell, M., Gustafsson, E., Aszódi, A., Morgelin,M., Bloch, W., Hunziker, E., Addicks, K., Timpl, R., and Fäqssler, R.(1999) J Cell Biol 147, 1109-1122).

Mature collagen fibres may contain several different types of boundaccessory proteins. They are part in the organisation of these fibresand regulate links to other molecules thereby contributing to thearchitecture of the fibrillar collagen network. A recent concept is thatof modulator molecules, which regulate the early steps in the assemblyof collagen monomers to fibres. Our laboratory has found that cartilageoligomeric matrix protein (COMP) accelerates the formation of fibresfrom monomers (Mörgelin and Heinegård, manuscript). Other molecules havethe opposite effect and slow down fibre formation in vitro, e.g. decorin(Vogel, K. G., Paulsson, M., and Heinegård, D. (1984) Biochem. J. 223,587-597) and fibromodulin (Hedborn, E. and Heinegård, D. (1989) J. Biol.Chem. 264, 6898-6905). Gene targeting of these molecules lead toabnormal collagen fibrils and disturbed mechanical properties of thetissues (Danielson, K. G., Baribault, H., Holmes, D. F., Graham, H.,Kadler, K. E., and Iozzo, R. V. (1997) J Cell Biol 136, 729-743;Svensson, L., Aszódi, A., Reinholt, F. P., Fäissler, R., Heinegård, D.,and Oldberg, Å. (1999) J. Biol. Chem. 274, 9636-9647). A picture isemerging where proteins in the vicinity of the cell regulate the earlystages of collagen fibre formation.

Perlecan exists as HS and CS substituted forms and it has been shownthat these forms can be used to facilitate collagen fibril formation. Toour surprise, the addition of free CS-E was effective in collagen fibrilformation, but none of the other CS variants had any significant effect(e.g. CS-D or CS-6).

A number of publications describing the effect of chondroitin sulphateon wound healing and for treating arthrosis exist (U.S. Pat. No.5,929,050, JP10120577 and RU2216332). The present invention differs fromthese significantly as the use of CS-E or active fragments thereofstimulates the formation of collagen based extracellular matrix (ECM)and thus acting as fibrillogenesis agonists or, by modification of CS-Eor active fragments thereof, as fibrosis antagonists.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the basic structure of chondroitin sulfate.

FIGS. 2A and 2B show graphs depicting data on CS-E accelerated collagenfibril formation, obtained from a collagen fibrillogenesis assay.

DESCRIPTION OF THE INVENTION

Comprised in the invention is the use of chondroitin sulphate fromPerlecan or chondroitin sulphate E (CS-E) or active fragments of CS-Efor treatment of various conditions and diseases related to disorders bythe facilitation or prevention of collagen fibril formation (CFF) byagonists and antagonists respectively.

Also comprised is a pharmaceutical acceptable composition containingCS-E or active fragments thereof for treating said conditions anddiseases.

CS-E or active fragments thereof are highly sulphated and thus may becharged compounds and the invention also comprises pharmaceuticalacceptable salts, such as alkali metal salts (sodium, potassium, cesium)and alkaline earth salts (e.g. magnesium, zinc, calcium, strontium) andammonium, as well as organic salts.

Also comprised in the invention is a formulation for administration of atherapeutically effective amount of CS-E, or active fragments thereof inproduction of artificial collagen matrices for treatment of disorders bytransplantation of cell containing or recruiting scaffolds.

Indications comprised in this application are conditions and diseasesrelated to disorders in collagen fibrillogenesis, including but notlimited to pulmonary fibrosis, wound healing, in particular chronicwound healing, chronic intestinal disease such as ulcerative colitis andCrohns disease, rheumatoid arthritis (RA), osteroarthritis (OA),reconstructive skeletal formation and skeletal repair.

CFF Agonists

Chronic wound healing can be stimulated by application of a CFF agonist,alone or together with exogenous collagen. This is also useful fortreatment of acute open wounds.

CFF agonists also facilitates healing of internal wounds including butnot limited to peptic ulcers, ulcerous colitis, Crohn's disease or otherinflammatory bowel disease.

CFF agonists can be used to facilitate healing after surgical treatmentand transplantation.

In the progressive degenerative joint disease osteoarthritis thearticular cartilage collagen is degraded. CFF agonists counteract orreverse this process and/or facilitates the assembly of newlysynthesized collagen molecules into fibers.

CFF agonists can be used to stimulate repair in damaged ligaments andtendons.

CFF agonist can be used to facilitate bone repair after severefractures.

CFF Agonists in Tissue Engineering

Tissue engineering can be used to repair damaged tissue (e.g. skin inburn wound healing or cartilage in OA) via autologous cell replacementtherapy or transplantation. CFF agonists can be used to stimulatecollagen matrix formation in cell or tissue culture for production oftransplantable skin, tendon, cartilage, bone or blood vessels frompatient cells or tissue or from various types of stem cells.

CFF agonists can be used to produce well-formed artificial collagenmatrix scaffolds for use in healing of e.g. burn wounds and OAcartilage. After implantation these scaffolds would be populated bycells recruited from the surrounding tissue of the patient.

CFF agonists are also useful in the production of well-organisedcollagen fibril matrices for corneal implants.

Fibrotic Disorders

Scarring is a natural response of the body to trauma and injury. Infibrotic conditions the normal wound healing response continues out ofcontrol, with excessive production and deposition of collagen. Thisleads to a loss of function when normal tissue is replaced with scartissue. Fibrosis can affect virtually all organ systems in the body.

There are many different causes of fibrosis, e.g. trauma, surgery,infection, environmental pollutants and toxins (including alcohol). Someexamples of fibrotic conditions are formation of scar tissue followingheart attack, kidney fibrosis as a complication of diabetes, lungfibrosis and surgical scar tissue formation between internal organs.

Acute fibrosis is a response to various forms of trauma, such as injury,infections, surgery, burns, radiation damage and chemotherapeutictreatments. Many chronic conditions, e.g. diabetes, viral infection andhypertension, induce a progressive fibrosis causing continuous loss oftissue function. The liver, kidney and lung are commonly affected.Systemic fibrotic diseases include diabetic nephropathy, scleroderma,idiopathic pulmonary fibrosis and reactive fibrosis following myocardialinfarct.

CFF Antagonists

By using CFF antagonists it will be possible to counteract fibroticprocesses by blocking the assembly of the collagen fibrils. In manycases this can be achieved by local administration thus avoidingpossible side effects from systemic treatment. However, the CFFantagonists may also be administered systemically when suitable.

CFF antagonists can be used to prevent fibrotic disorders of the skin,including, but not limited to scar formation in wound healing,hypertrophic scarring and keloid, contracture in connection withhypertrophic scarring after burn injury, surgical adherens orskleroderma. Local application of the CFF antagonists would be easy,except in surgical adherens where administration could be achieved byosmotic pump devices or other suitable administrations.

CFF antagonists can be used for treatment and prevention of idiopathiclung fibrosis.

CFF antagonists could be used to treat other deep organ fibrosis such asliver fibrosis/cirrhosis and diabetic kidney fibrosis.

CFF antagonists could be used to prevent heart muscle scarring aftermyocardial infarction.

CFF antagonists could be used to prevent or counteract atherosclerosisand restenosis after angioplasty. To achieve the latter, the antagonistcould be delivered locally by implantation of modified stents.

EXAMPLES In Vitro Fibrillogenesis Assay

Bovine pepsin-extracted collagen type I was purchased from Vitrogen.Collagen II was pepsin extracted from bovine tracheal cartilage, aspreviously described (Vogel, K. G., Paulsson, M., and Heinegård, D.(1984) Biochem. J. 223, 587-597). The fibrillogenesis assay has beenpreviously described (Hedborn, E. and Heinegård, D. (1989) J. Biol.Chem. 264, 6898-6905).

Briefly, a solution of collagen monomers (330 nM) was brought to neutralpH by addition of an appropriate volume of 0.012M NaOH, and buffered by20 mM HEPES, 150 mM NaCl at pH 7.4. Perlecan fragment was added atconcentrations equimolar to that of collagen or at one tenth the molarconcentration of collagen. The sample was mixed vigorously and briefly,and transferred to a cuvette. The sample was incubated at 37° C.(collagen type I) or 35° C. (collagen type II) in water-jacketedcuvettes in the spectrophotometer, and the absorbance due to lightscattering at 400 nm (collagen type I) or 313 nm (collagen type II) wasmonitored over a duration of 5-18 hrs. The increasedabsorbance/turbidity depends on increasing fibre formation.

CS-E Fibrillogenesis

Fibrillogenesis was performed as described above. CS-E from squid waspurchased from Calbiochem. 0.13 μg/ml corresponding to the molarconcentration of GAG chains in previous experiments using recombinantperlecan domain I variants with HS/CS (PG IB) (33 nM).

The results of the fibrillogenesis experiment is shown in FIG. 2. As canbe seen from this figure, CS-E but none of the other tested chondroitinsulphate variants (CS-D and CS-6) gives a positive result on collagenfibril formation (CFF).

FIG. 2 CS-E accelerated collagen fibril formation. Different types ofpurified CS-chains were tested for effect in the collagenfibrillogenesis assay (panel A). The highly sulphated CS-E had dramaticeffects on fibril formation but neither CS-6 nor CS-D had any effect.The stimulatory effect of CS-E was dose-dependent, reaching saturationat concentration of 30 μg/ml (panel B).

In Vivo Model

Example of In Vivo Model for Studying Wound Healing.

Groups of 5 ICR male mice weighing 22±2 gms are used. Under hexobarbital(90 mg/kg, i.p.) anesthesia, the shoulder and back region of each animalis shaved. A sharp punch (ID 12 mm) is used to remove the skin includingpanniculus carnosus and adherent tissues. The wound area, traced ontoclear plastic sheets on days 3, 5, 7, 9 and 11, are quantitated by useof an Image Analyzer (Life Science Resources VISTA, Version 3.0). Testcompound and/or vehicle (20 μl, 0.5% carboxymethylcellulose in PBS pH7.4) is applied topically immediately following injury and once dailythereafter for a total of 10 consecutive days. The wound half closuretime (CT₅₀) is determined and unpaired Student's t-test is applied forcomparison between treated and vehicle group at each measurement timepoint. Differences are considered statistical significance at P<0.05.(Montesinos, M. C., Gadangi, P., Longaker, M., Sung, J., Levine, J.,Nilsen, D., Reibman, J., Li, M., Jiang, C. K., Hirschom, R., Recht, P.A., Ostad, E., Levin, R. I. and Crostein, B. N. Wound healing isaccelerated by agonists of Adenosine A₂ (Gα_(s)-linked) receptors. J.Exp. Med. 186: 1615-1620, 1997.)

Formulation

Example of a Preparation Comprising a Capsule

Per capsule Active ingredient, as salt 5 mg Lactose 250 mg Starch 120 mgMagnesium stearate 5 mg Total up to 385 mg

In case higher amounts of active ingredient are required, the amount oflactose used may be reduced.

Example of a Suitable Tablet Formulation.

Per tablet Active ingredient, as salt 5 mg Potato starch 90 mg Colloidalsilica 10 mg Talc 20 mg Magnesium stearate 2 mg 5% aqueous solution ofgelatine 25 mg Total up to 385 mg

A solution for parenteral administration by injection can be prepared inaqueous solution of a water-soluble pharmaceutically acceptable acidaddition salt of the active substance preferably in a concentration of0.1% to about 10% by weight.

These solutions may also contain stabilising agents, buffering agentsand/or gelating agents such as but not limited to hyaluronan, PEG, HPMC,EHEC, to obtain a controlled release and/or elimination.

Example of a Topical Formulation

A gel for topical administration can be prepared an active substance ina concentration of 0.1% to 10% by weight, optionally containingstabilising agents, buffering agents and/or additional gelating agentssuch as but not limited to hyaluronan, PEG, HMPC, EHEC to obtaincontrolled release and/or elimination

1. A method of facilitating collagen fibril formation in a wound in asubject, the method comprising administering to said subject aneffective amount of chondroitin sulphate E, wherein the chondroitinsulphate E is administered at a concentration of between 0.13 μg/ml and60 μg/ml.
 2. A method as claimed in claim 1, wherein the chondroitinsulphate E is administered at a concentration of between 0.13 μg/ml and30 μg/ml.
 3. A method as claimed in claim 2, wherein the chondroitinsulphate E is administered at a concentration of between 57.8 μg/ml and30 μg/ml.
 4. A method as claimed in claim 1, wherein the effectiveamount of chondroitin sulphate E is administered by oral, topical, orinjectable route.
 5. A method as claimed in claim 4, wherein theeffective amount of chondroitin sulphate E is administered by topicalroute.
 6. A method as claimed in claim 5, wherein the chondroitinsulphate E is administered in a gel.
 7. A method as claimed in claim 4,wherein the chondroitin sulphate E is administered by an oral route. 8.A method as claimed in claim 1, wherein said chondroitin sulphate E ischondroitin sulphate E obtained from squid.
 9. A method as claimed inclaim 1, wherein a pharmaceutical composition comprising purifiedchondroitin sulphate E is administered.
 10. A method of facilitatingcollagen fibril formation in an ulcer in a subject, the methodcomprising administering to said subject an effective amount ofchondroitin sulphate E, wherein the chondroitin sulphate E isadministered at a concentration of between 0.13 μg/ml and 60 μg/ml. 11.A method as claimed in claim 10, wherein the chondroitin sulphate E isadministered at a concentration of between 0.13 μg/ml and 30 μg/ml. 12.A method as claimed in claim 11, wherein the chondroitin sulphate E isadministered at a concentration of between 7.8 μg/ml and 30 μg/ml.
 13. Amethod as claimed in claim 10, wherein the effective amount ofchondroitin sulphate E is administered by oral, topical or injectableroute.
 14. A method as claimed in claim 13, wherein the effective amountof chondroitin sulphate E is administered by topical route.
 15. A methodas claimed in claim 14, wherein the chondroitin sulphate E isadministered in a gel.
 16. A method as claimed in claim 13, wherein thechondroitin sulphate E is administered by an oral route.
 17. A method asclaimed in claim 10, wherein said chondroitin sulphate E is chondroitinsulphate E obtained from squid.
 18. A method as claimed in claim 10,wherein a pharmaceutical composition comprising purified chondroitinsulphate E is administered.